Sample Preparation Research Articles

A Comprehensive Review on Capillary Electrophoresis-Mass Spectrometry in Advancing Biomolecular Research.

This review provides an in-depth exploration of capillary electrophoresis-mass spectrometry (CE-MS) in biomolecular research from 2020 to 2024. CE-MS emerges as a versatile and powerful tool due to its numerous advantages, facilitating the analysis of various biomolecules, including proteins, peptides, oligonucleotides, and other metabolites, such as lipids, carbohydrates, or amines, among others. The review extends to various CE modes and interfaces for the CE-MS coupling, offering comprehensive insights into their applications within biomolecular research. Furthermore, it effectively summarizes the conditions employed in CE-MS while also addressing critical aspects such as sample preparation requirements. Despite its advantages, the review highlights a gap between discovery and practical implementation, underscoring the need for large-scale validation and method standardization to fully realize the potential of CE-MS in biomolecular research.

Metabolomics and proteomics insights into hepatic responses of weaned piglets to dietary Spirulina inclusion and lysozyme supplementation.

Studying the effect of dietary Spirulina and lysozyme supplementation on the metabolome and proteome of liver tissue contributes to understanding potential hepatic adaptations of piglets to these novel diets. This study aimed to understand the influence of including 10% Spirulina on the metabolome and proteome of piglet liver tissue. Three groups of 10 post-weaned piglets, housed in pairs, were fed for 28days with one of three experimental diets: a cereal and soybean meal-based diet (Control), a base diet with 10% Spirulina (SP), and an SP diet supplemented with 0.01% lysozyme (SP + L). At the end of the trial, animals were sacrificed and liver tissue was collected. Metabolomics analysis (n = 10) was performed using NMR data analysed with PCA and PLS-DA. Proteomics analysis (n = 5) was conducted using a filter aided sample preparation (FASP) protocol and Tandem Mass Tag (TMT)-based quantitative approach with an Orbitrap mass spectrometer. Growth performance showed an average daily gain reduction of 9.5% and a feed conversion ratio increase of 10.6% in groups fed Spirulina compared to the control group. Metabolomic analysis revealed no significant differences among the groups and identified 60 metabolites in the liver tissue. Proteomics analysis identified 2,560 proteins, with 132, 11, and 52 differentially expressed in the Control vs. SP, Control vs. SP + L and SP vs. SP + L comparisons, respectively. This study demonstrated that Spirulina enhances liver energy conversion efficiency, detoxification and cellular secretion. It improves hepatic metabolic efficiency through alterations in fatty acid oxidation (e.g., upregulation of enzymes like fatty acid synthase and increased acetyl-CoA levels), carbohydrate catabolism (e.g., increased glucose and glucose-6-phosphate), pyruvate metabolism (e.g., higher levels of pyruvate and phosphoenolpyruvate carboxykinase), and cellular defence mechanisms (e.g., upregulation of glutathione and metallothionein). Lysozyme supplementation mitigates some adverse effects of Spirulina, bringing physiological responses closer to control levels. This includes fewer differentially expressed proteins and improved dry matter, organic matter and energy digestibility. Lysozyme also enhances coenzyme availability, skeletal myofibril assembly, actin-mediated cell contraction, tissue regeneration and development through mesenchymal migration and nucleic acid synthesis pathways. While Spirulina inclusion had some adverse effects on growth performance, it also enhanced hepatic metabolic efficiency by improving fatty acid oxidation, carbohydrate catabolism and cellular defence mechanisms. The addition of lysozyme further improved these benefits by reducing some of the negative impacts on growth and enhancing nutrient digestibility, tissue regeneration, and overall metabolic balance. Together, Spirulina and lysozyme demonstrate potential as functional dietary components, but further optimization is needed to fully realize their benefits without compromising growth performance.

In situ isotropic 3D imaging of vasculature perfusion specimens using x-ray microscopic dual-energy CT.

Ex vivo x-ray angiography provides high-resolution, three-dimensional information on vascular phenotypes down to the level of capillaries. Sample preparation for ex vivo angiography starts with the removal of blood from the vascular system, followed by perfusion with an x-ray dense contrast agent mixed with a carrier such as gelatine or a polymer. Subsequently, the vascular micro-architecture of harvested organs is imaged in the intact fixed organ. In the present study, we present novel microscopic dual-energy CT (microDECT) imaging protocols that allow to visualise and analyse microvasculature in situ with reference to the morphology of hard and soft tissue. We show that the spectral contrast of µAngiofil and Micropaque barium sulphate in perfused specimens allows for the effective separation of vasculature from mineralised skeletal tissues. Furthermore, we demonstrate the counterstaining of perfused specimens using established x-ray dense contrast agents to depict blood vessels together with the morphology of soft tissue. Phosphotungstic acid (PTA) is used as a counterstain that shows excellent spectral contrast in both µAngiofil and Micropaque barium sulphate-perfused specimens. A novel Sorensen-buffered PTA protocol is introduced as a counterstain for µAngiofil specimens, as the polyurethane polymer is susceptible to artefacts when using conventional staining solutions. Finally, we demonstrate that counterstained samples can be automatically processed into three separate image channels (skeletal tissue, vasculature and stained soft tissue), which offers multiple new options for data analysis. The presented microDECT workflows are suited as tools to screen and quantify microvasculature and can be implemented in various correlative imaging pipelines to target regions of interest for downstream light microscopic investigation.

Native Cellular Membranes Facilitate Channel Activity of MscL by Enhancing Slow Collective Motions of Its Transmembrane Helices.

Mechanosensitive channels of large conductance (MscL) serve as a mechanoelectrical valve of cells in response to the membrane tension. The influence of membrane environments on the MscL channel activity and the underlying mechanism remains unclear. Herein, we developed a new sample preparation protocol that allows for the detection of high-quality 1H-detected solid-state NMR spectra of MscL in cellular membranes, enabling site-specific analysis of its dynamics. Dipolar order parameters and spin relaxation rates are measured for 51 residues of MscL in synthetic and native membranes. The dynamics data reveal that while MscL maintains a similar rigidity in both membrane environments, it exhibits enhanced slow collective motions in the native cellular membranes. Molecular dynamics simulations demonstrate the critical role of slow motions in the mechanosensitivity of MscL by promoting protein-membrane interactions. This study examines atomic-resolution dynamics of a membrane-protein in cellular membranes and provides novel insights into the functional significance of membrane-protein dynamics.

Recent Progress and Applications of Advanced Nanomaterials in Solid-Phase Extraction.

Sample preparation maintains a key bottleneck in the whole analytical procedure. Solid-phase sorbents (SPSs) have garnered increasing attention in sample preparation research due to their crucial roles in achieving high clean-up and enrichment efficiency in the analysis of trace targets present in complex matrices. Novel nanoscale materials with improved characteristics have garnered considerable interest across different scientific disciplines due to the limited capabilities of traditional bulk-scale materials. The purpose of this review is to offer a thorough summary of the latest developments and uses of SPSs in preparing samples for chromatographic analysis, focusing on the years 2020-2024. The techniques for preparing SPSs are examined, such as metal-organic frameworks (MOFs), covalent organic frameworks (COFs), carbon nanoparticles (CNPs), molecularly imprinted polymers (MIPs), and metallic nanomaterials (MNs). Examining the pros and cons of different extraction methods, including solid-phase extraction (SPE), magnetic SPE (MSPE), flow-based SPE (FBA-SPE), solid-phase microextraction (SPME), stir-bar sorptive extraction (SBSE), and dispersive SPE (DSPE), is the main focus. Furthermore, this article presents the utilization of SPE technology for isolating common contaminants in various environmental, biological, and food specimens. We highlight the persistent challenges in SPSs and anticipate future advancements and applications of novel SPSs.

Anthropogenic Impact on Soils of Cities Irkutsk, Angarsk

The morphological field description of soil profiles of the territory of Irkutsk region was carried out. Samples of soils, soil-forming rocks and vegetation were taken for further physical and chemical analyses to determine soil properties in the studied areas. Sample preparation of soils was carried out, phytotoxicity of soils was analyzed. Processing of the obtained data was carried out, as a result of which the percentage of germination of radish seeds in the upper horizons of soils of the key areas of Irkutsk and Angarsk cities was revealed, the length of shoots and the length of roots in the soils of the studied samples were measured, the index of soil toxicity was calculated.

Electroanalysis of Statin Drugs: A Review on the Electrochemical Sensor Architectures Ranging from Classical to Modern Systems.

An overview of the latest advances in the design of electrochemical sensor architectures dedicated to the determination of drugs from the statin class is presented in this review. Statins are drugs widely consumed for cholesterol control, and their determination in different matrices through the application of electroanalysis is growing considering advantages such as operational simplicity, lower cost and ease of sample preparation. Within the context of statins, electrochemical sensor architectures can be subdivided into conventional/classical electrodes such as glassy carbon electrodes, carbon paste electrodes, pencil graphite electrodes, boron-doped diamond electrodes and metallic electrodes, and more modern electrode systems, including the screen-printed electrodes and 3D-printed electrodes. Thus, different aspects related to the preparation of these electrochemical sensors and analytical performance are presented, also reflecting advances in terms of designs of new architectures and possible improvements not previously reviewed. Analyzed samples, advantages and disadvantages of different implemented sensor's modification strategies and perspectives for the electroanalysis of statins are also included throughout the work.

Dynamic Magnetic Susceptibility Method in Studies of Coordination Compounds

The measurement of the dynamic magnetic susceptibility is a universal method, which is used for the evaluation of magnetic properties of single molecule magnets by scientists all over the world. An information in the Russian scientific literature that can be useful for practical mastering of this method is presently insufficient. To fill this gap, in this work we present a detailed procedure of a magnetochemical experiment for observing slow magnetic relaxation in coordination compounds of 3d- and 4f-element ions and the complete characterization of the dynamics of the magnetic behavior. Special attention is given to usually omitted but important details related to all stages of studying the magnetic relaxation dynamics. The main variants of sample preparation are described, the logics of the construction of a measuring sequence and the procedure of experimental data processing are discussed, and advantages and drawbacks of some programs of the calculation of magnetic relaxation dynamics data are considered. The main concepts and equations used in experimental data analysis are presented, and the primary conclusions that can be made from the obtained results are proposed.

Fully Integrated Microfluidic Digital Chip for Simple and Highly Quantitative Detection of Norovirus.

The prevalence of foodborne illnesses is a significant global concern, resulting in numerous illnesses, deaths, and substantial economic losses annually. Traditional detection methods for foodborne pathogens are often slow, limited, and impractical for field use, underscoring the need for rapid, sensitive, and portable assays. Microfluidic technology has emerged as a promising solution for sample preparation, reaction, and detection on a small scale. Our study introduces a novel microfluidic digital loop-mediated isothermal amplification (LAMP) assay platform, which employs digital microfluidic chips for absolute quantitative analysis of nucleic acids. This portable chip utilizes LAMP technology to achieve ultrasensitive detection of target nucleic acids within 30 min and reduces the detection limit to 1 fM without the need for complex instrumentation. By digitizing amplification signals directly from the target sample, our platform offers simplicity, affordability, portability, and quantitative molecular readouts. This innovation represents a crucial step toward the on-site detection of foodborne pathogens, thereby enhancing food safety and mitigating disease outbreaks.

Farm to fork applications: how vibrational spectroscopy can be used along the whole value chain?

Vibrational spectroscopy is a nondestructive analysis technique that depends on the periodic variations in dipole moments and polarizabilities resulting from the molecular vibrations of molecules/atoms. These methods have important advantages over conventional analytical techniques, including (a) their simplicity in terms of implementation and operation, (b) their adaptability to on-line and on-farm applications, (c) making measurement in a few minutes, and (d) the absence of dangerous solvents throughout sample preparation or measurement. Food safety is a concept that requires the assurance that food is free from any physical, chemical, or biological hazards at all stages, from farm to fork. Continuous monitoring should be provided in order to guarantee the safety of the food. Regarding their advantages, vibrational spectroscopic methods, such as Fourier-transform infrared (FTIR), near-infrared (NIR), and Raman spectroscopy, are considered reliable and rapid techniques to track food safety- and food authenticity-related issues throughout the food chain. Furthermore, coupling spectral data with chemometric approaches also enables the discrimination of samples with different kinds of food safety-related hazards. This review deals with the recent application of vibrational spectroscopic techniques to monitor various hazards related to various foods, including crops, fruits, vegetables, milk, dairy products, meat, seafood, and poultry, throughout harvesting, transportation, processing, distribution, and storage.

Membrane-based preparation for mass spectrometry imaging of cultures of bacteria.

The study of the dialogue between microorganisms at the molecular level is becoming essential to understand their relationship (antagonist, neutral, or beneficial interactions) and its impact on the organization of the microbial community. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption/ionization (MALDI) is a technique that reveals the spatial distribution of molecules on a sample surface that may be involved in interactions between organisms. An experimental limitation to perform MALDI MSI is a flat sample surface, which in many cases could not be achieved for bacterial colonies such as filamentous bacteria (e.g., Streptomyces). In addition, sample heterogeneity affects sample dryness and MALDI matrix deposition prior to MSI. To avoid such problems, we introduce an additional step in the sample preparation. A polymeric membrane is interposed between the microorganisms and the agar-based culture medium, allowing the removal of bacterial colonies prior to MSI of the homogeneous culture medium. A proof of concept was evaluated on Streptomyces ambofaciens (a soil bacterium) cultures on solid media. As the mycelium was removed at the same time as the polymeric membrane, the metabolites released into the medium were spatially resolved by MALDI MSI. In addition, extraction of the recovered mycelium from the membrane confirmed the identification of the metabolites by ESI MS/MS analysis. This approach allows both the spatial distribution of metabolites produced by microorganisms in an agar medium to be studied under well-controlled sample preparation and their structure to be elucidated. This capability is illustrated using desferrioxamine E, a siderophore produced by S. ambofaciens.

Experimental determination of the coefficient of viscosity of the dry snow

Snow compaction is a natural process in the evolution of any snow cover, and this obviously changes the physical and mechanical properties of a snow thickness. The quantitative estimate of this process is a coefficient of the snow viscosity, which is linearly related to the rate of the snow cover deformation The technique for preparation and testing of samples with the fine-grain snow under condition of uniaxial compression at negative temperatures is described. It was determined that values of the snow viscosity coefficient fall within the range from 0.10 to 0.30 g/cm³ for three temperature intervals. The viscosity coefficient of the fine-grain snow varies from 10⁸ до 10¹⁰ Па. The data obtained made possible to find the limits of variability of this coefficient for winter conditions on the Russian plain. A dependence of the viscosity coefficient on the snow density and temperature is proposed, and it describes the series of the above shown experimental data. It is shown that influence of changes in the viscosity coefficient of fine-grain snow on the snow properties depending on its density are comparable in scale to the influence of the temperature. The result may be useful for modeling the evolution of snow cover.

Versatility of Raman spectroscopy for studies on the back-end of the nuclear fuel cycle

AbstractIn recent years, Raman spectroscopy has been proven to be a highly versatile characterization technique for nuclear materials research. This sensitive technique possesses, among others, two relevant features that comply with the safety conditions required when handling nuclear materials: its flexibility allows for remote, in situ and ex situ analysis, and its relative ease of use implies small sample quantities and limited effort for sample preparation. In this work, we present the acquisition protocol and data processing necessary for obtaining important information within the back-end of the nuclear fuel cycle. Specifically, we focus on research studies on the advanced characterization of nuclear fuels and the development of hydrometallurgical separation processes. The results described here were obtained by using different nuclear fuels analogs, but the acquisition protocol and data processing described can be applied to the real nuclear fuel cycle. Graphical abstract

Enhancing silicon spectral emission in LIBS using Tesla coil discharge

Laser-induced breakdown spectroscopy (LIBS) is a powerful technique for elemental analysis, offering rapid analysis, minimal sample preparation, wide elemental coverage, and portability. To enhance the detection sensitivity of LIBS, increasing the spectral emission intensity is crucial. This paper explores the use of Tesla coil (TC) discharge as an alternative to spark discharge in silicon LIBS. The study examines the influence of TC discharge on both time-integrated and time-resolved spectra, with and without TC discharge; the corresponding electron temperature and density are obtained. The results show that TC discharge significantly amplifies the spectral intensity, improving signal sensitivity in LIBS analysis. Specifically, in the laser energy range from 7.4 to 24.0 mJ, TC discharge increased the average spectral line intensities of Si (II) 385.60 nm and Si (I) 390.55 nm by factors of 8.4 and 5.1, respectively. Additionally, the average electron temperature and density were enhanced by approximately 3.2% and 4.2%, respectively, under TC discharge. The advantages of TC discharge include higher energy deposition, extended discharge duration, reduced electrode erosion, and enhanced safety. This research contributes to advancing LIBS technology and expanding its applications in various fields.

A Simple and Green Analytical Alternative for Chloride Determination in High-Salt-Content Crude Oil: Combining Miniaturized Extraction with Portable Colorimetric Analysis

A simple and miniaturized protocol was developed for chloride extraction from Brazilian pre-salt crude oil for further salt determination by colorimetry. In this protocol, the colorimetric analysis of chloride using digital images was carried out in an aqueous phase obtained after a simple and miniaturized extraction carefully developed for this purpose. A portable device composed of a homemade 3D-printed chamber with a USB camera was used. The PhotoMetrix app converted the images into RGB histograms, and a partial least squares (PLS) model was obtained from chemometric treatment. The sample preparation was performed by extraction after defining the best conditions for the main parameters (e.g., extraction time, temperature, type and volume of solvent, and sample mass). The PLS model was evaluated considering the coefficient of determination (R2) and the root mean square errors (RMSEs)—calibration (RMSEC), cross-validation (RMSECV), and prediction (RMSEP). Under the optimized conditions, an extraction efficiency higher than 84% was achieved, and the limit of quantification was 1.6 mg g−1. The chloride content obtained in the pre-salt crude oils ranged from 3 to 15 mg g−1, and no differences (ANOVA, 95%) were observed between the results and the reference values by direct solid sampling elemental analysis (DSS-EA) or the ASTM D 6470 standard method. The easy-to-use colorimetric analysis combined with the extraction method’s simplicity offered a high-throughput, low-cost, and environmentally friendly method, with the possibility of portability. Additionally, the decrease in energy consumption and waste generation, increasing the sample throughput and operators’ safety, makes the proposed method a greener approach. Furthermore, the cost savings make this a suitable option for routine quality control, which can be attractive in the crude oil industry.

Identification of hypoxene metabolites in urine samples using gas chromatography–tandem mass spectrometry for anti-doping control

Objectives. Hypoxen is a drug which possesses antioxidant and antihypoxic effects. It achieves this by increasing the utilization of oxygen by mitochondria, intensifying oxidative phosphorylation, and as a result, improving tissue respiration. Athletes take it during prolonged exercise, in order to increase efficiency and reduce physical overwork. Since 2023, the World Anti-Doping Agency has included the drug in the monitoring program, in the belief that it can be used to gain a competitive advantage. It is thus a candidate for inclusion in the Prohibited List as a potential regulator of the human metabolism. Currently, there are no studies or scientific publications focusing on the identification of hypoxene in biofluids for the purpose of anti-doping control. The aim of this study is to determine the possible metabolites of the drug and their chromato-mass spectrometric characteristics in urine samples using gas chromatography–tandem mass spectrometry (GC–MS/MS) for doping control screening purposes.Methods. Sample preparation of urine samples was carried out using enzymatic hydrolysis, liquid–liquid extraction and derivatization. The GC–MS/MS method was used for analysis. Screening of hypoxene metabolites was carried out in the mode of total ion current after fragmentation of selected parent ions.Results. Three specific metabolites of hypoxene (m/z 342, 300, and 346, including trimethylsilyl derivatives) were identified in urine samples of volunteers (n = 3). They can act as markers for taking the target antihypoxant, and their possible structural formulas are given. The excretion curves of two metabolites with an m/z of 300 and 346 respectively in urine were studied. The maximum concentration is reached after 8–14 and 1.5–6 h, respectively. It was established, that these metabolites are reliably identified in urine 90 h or more after a single dose of the drug.Conclusions. Possible structures of hypoxene metabolites in urine samples from volunteers were determined for the first time and their chromato-mass spectrometric characteristics were established. The approach developed in this study can be used for screening analysis of hypoxene for the purpose of anti-doping control.

Analytical Determination of Squalene in Extra Virgin Olive Oil and Olive Processing By-Products, and Its Valorization as an Ingredient in Functional Food—A Critical Review

Squalene is a bioactive compound with significant health benefits, predominantly found in extra virgin olive oil (EVOO) and its processing by-products. This critical review explores the analytical determination of squalene in EVOO and various by-products from olive oil production, highlighting its potential as a valuable ingredient in functional foods. An overview of existing analytical methods is provided, focusing on different approaches to sample preparation before analytical determination, evaluating their effectiveness in quantifying squalene concentrations. Studies not primarily centered on analytical methodologies or squalene quantification were excluded. A critical gap identified is the absence of an official method for squalene determination, which hinders comparability and standardization across studies, underscoring the importance of developing a reliable, standardized method to ensure accurate quantification. The valorization of squalene involves advocating for its extraction from olive oil processing by-products to enhance sustainability in the olive oil industry. By recovering squalene, the industry can not only reduce waste but also enhance functional food products with this health-promoting compound. Additionally, there is a need for economically sustainable and environmentally friendly extraction techniques that can be scaled up for industrial application, thus contributing to a circular economy within the olive oil sector.

On-site extraction of phenoxycarboxylic acid herbicides in environmental waters utilizing monolith-based in-tip microextraction technique

On-site extraction plays a significant role in the reliable quantification of strong polar phenoxycarboxylic acid herbicides (PCAs) in aqueous samples. In current study, a new technique for the field sample preparation of PCAs was developed by means of three channels in-tip microextraction device (TCIM). To capture PCAs effectively, an extraction phase based on monolith (EPM) using vinylimidazole and divinylbenzene/ethylene dimethacrylate as monomer and cross-linkers, respectively, was in-situ synthesized in pipette tips. The EPM fabricated at optimal conditions were characterized by a series of techniques and employed as the adsorbent of TCIM for the on-site extraction of PCAs. The adsorption isotherm was studied so as to inspect the extraction behaviors of EPM towards PCAs. Results revealed that the proposed EPM/TCIM presented satisfactory extraction performance towards PCAs through multiple interactions. The enrichment factors and adsorption capacity were 74-277 and 20 mg/g, respectively. Under the most beneficial extraction parameters, the developed EPM/TCIM was successfully employed to on-site extract PCAs, and then combining with HPLC equipped with diode array detector to monitor trace PCAs in actual waters. The limits of detection (LODs) towards investigated PCAs varied from 0.071 μg/L to 0.30 μg/L. In addition, the accuracy of established approach was inspected with documented method. Compared with existing lab-based sample preparation approaches, the introduced field sample preparation technique exhibits some merits such as avoidance of transporting large volume of water, prevention of analytes loss during sampling procedure, less usage of organic solvent and achievement of satisfactory efficient in sample preparation.

Development and validation of an LC-MS/MS method for highly concentrated tacrolimus and cyclosporine samples prepared from pharmaceutical products to assess drug loss from feeding tubes

IntroductionTacrolimus and cyclosporine are common immunosuppressants utilized post-organ transplantation to manage allograft rejection. Both have narrow therapeutic indices and are frequently measured to support dose adjustments. Although nasogastric tubes are commonly used to provide nutritional support and serve as a route for immunosuppressant administration, they were never validated for such purposes. ObjectiveTo develop and validate a liquid chromatography – tandem mass spectrometry (LC-MS/MS) method for highly concentrated tacrolimus and cyclosporine samples prepared from pharmaceutical products to support the validation of feeding tube administration of these immunosuppressants. MethodsThe method involved stepwise dilutions with dimethyl sulfoxide before analysis using online sample preparation and LC-MS/MS. It was validated in a CLIA-certified clinical laboratory that measures immunosuppressants by LC-MS/MS and is designed to support clinical studies evaluating drug loss from feeding tubes. ResultsThe method was linear between 6.8 µg/mL and 75 µg/mL for tacrolimus, and between 0.9 mg/mL and 10 mg/mL for cyclosporine, with r2 > 0.99 and total precision <5 % at all QC levels. The method demonstrated good recovery using certified cyclosporine reference material, European pharmacopeia reference standard tacrolimus, and prepared pharmaceutical products. Minimal matrix effects were observed. ConclusionAn analytical method was developed and validated for in vitro studies with simulated administration of tacrolimus or cyclosporine to assess loss during drug administration using feeding tubes.

Ionic liquids-based adsorbents for extraction and separation: A review emphasizing recent advances in sample preparation

Ionic liquids-based adsorbents for extraction and separation: A review emphasizing recent advances in sample preparation